Alterations in topoisomerase IV and DNA gyrase in quinolone-resistant mutants of Mycoplasma hominis obtained in vitro

Mycoplasma hominis mutants were selected stepwise for resistance to ofloxacin and sparfloxacin, and their gyrA, gyrB, parC, and parE quinolone resistance-determining regions were characterized. For ofloxacin, four rounds of selection yielded six first-, six second-, five third-, and two fourth-step mutants. The first-step mutants harbored a single Asp426-->Asn substitution in ParE. GyrA changes (Ser83-->Leu or Trp) were found only from the third round of selection. With sparfloxacin, three rounds of selection generated 4 first-, 7 second-, and 10 third-step mutants. In contrast to ofloxacin resistance, GyrA mutations (Ser83-->Leu or Ser84-->Trp) were detected in the first-step mutants prior to ParC changes (Glu84-->Lys), which appeared only after the second round of selection. Further analysis of eight multistep-selected mutants of M. hominis that were previously described (2) revealed that they carried mutations in ParE (Asp426-->Asn), GyrA (Ser83-->Leu) and ParE (Asp426-->Asn), GyrA (Ser83-->Leu) and ParC (Ser80-->Ile), or ParC (Ser80-->Ile) alone, depending on the fluoroquinolone used for selection, i.e., ciprofloxacin, norfloxacin, ofloxacin, or pefloxacin, respectively. These data indicate that in M. hominis DNA gyrase is the primary target of sparfloxacin whereas topoisomerase IV is the primary target of pefloxacin, ofloxacin, and ciprofloxacin.     

Sparfloxacin resistance in clinical isolates of Streptococcus pneumoniae: involvement of multiple mutations in gyrA and parC genes

Antimicrobial susceptibility testing revealed among 150 clinical isolates of Streptococcus pneumoniae 4 pneumococcal isolates with resistance to fluoroquinolones (MIC of ciprofloxacin, >/=32 microgram/ml; MIC of sparfloxacin, >/=16 microgram/ml). Gene amplification and sequencing analysis of gyrA and parC revealed nucleotide changes leading to amino acid substitutions in both GyrA and ParC of all four fluoroquinolone-resistant isolates. In the case of strains 182 and 674 for which sparfloxacin MICs were 16 and 64 microgram/ml, respectively, nucleotide changes were detected at codon 81 in gyrA and codon 79 in parC; these changes led to an Ser-->Phe substitution in GyrA and an Ser-->Phe substitution in ParC. Strains 354 and 252, for which sparfloxacin MICs were 128 microgram/ml, revealed multiple mutations in both gyrA and parC. These strains exhibited nucleotide changes at codon 85 leading to a Glu-->Lys substitution in GyrA, in addition to Ser-79-->Tyr and Lys-137-->Asn substitutions in ParC. Moreover, strain 252 showed additional nucleotide changes at codon 93, which led to a Trp-->Arg substitution in GyrA. These results suggest that sparfloxacin resistance could be due to the multiple mutations in GyrA and ParC. However, it is possible that other yet unidentified mutations may also be involved in the high-level resistance to fluoroquinolones in S. pneumoniae.     

Primary targets of fluoroquinolones in Streptococcus pneumoniae

Mutants of wild-type Streptococcus pneumoniae IID553 with mutations in parC were obtained by selection with trovafloxacin, levofloxacin, norfloxacin, and ciprofloxacin. All of the parC mutants were cross-resistant to the selecting agents but were not resistant to gatifloxacin and sparfloxacin. On the other hand, gyrA mutants were isolated by selection with gatifloxacin and sparfloxacin. The gyrA mutants were cross-resistant to gatifloxacin and sparfloxacin but were not resistant to the other fluoroquinolones tested. These results suggest that in wild-type S. pneumoniae the primary target of trovafloxacin, levofloxacin, norfloxacin, and ciprofloxacin is topoisomerase IV, whereas the primary target of gatifloxacin and sparfloxacin is DNA gyrase.     

ParC and GyrA may be interchangeable initial targets of some fluoroquinolones in Streptococcus pneumoniae

To evaluate the role of known topoisomerase IV and gyrase mutations in the fluoroquinolone (FQ) resistance of Streptococcus pneumoniae, we transformed susceptible strain R6 with PCR-generated fragments encompassing the quinolone resistance-determining regions (QRDRs) of parC or gyrA from different recently characterized FQ-resistant mutants. Considering the MICs of FQs and the GyrA and/or ParC mutations of the individual transformants, we found three levels of resistance. The first level was obtained when a single target, ParC or GyrA, depending on the FQ, was modified. An additional mutation(s) in a second target, GyrA or ParC, led to the second level. The highest increases in resistance levels were seen for Bay y3118 and moxifloxacin with the transformant harboring a double mutation in both ParC and GyrA. When a single modified target was considered, only the ParC mutation(s) led to an increase in the MICs of pefloxacin and trovafloxacin. In contrast, the GyrA or ParC mutation(s) could lead to increases in the MICs of ciprofloxacin, sparfloxacin, grepafloxacin, Bay y3118, and moxifloxacin. These results suggest that the preferential target of trovafloxacin and pefloxacin is ParC, whereas either ParC or GyrA may both be initial targets for the remaining FQs tested. The contribution of the ParC and GyrA mutations to efflux-mediated FQ resistance was also examined. Active efflux was responsible for two- to fourfold increases in the MICs of ciprofloxacin for the transformants, regardless of the initial FQ resistance levels of the recipients.     

Isolation and characterization of a dibenzofuran-degrading yeast: identification of oxidation and ring cleavage products

PURPOSE: To investigate the development of fluoroquinolone resistance among Neisseria gonorrhoeae isolates in Japan and the frequency and patterns of mutations involving the GyrA and ParC proteins, which confer quinolone resistance to the bacteria, in isolates. MATERIALS AND METHODS: Antimicrobial susceptibilities of 145 gonococcal isolates, including 79 isolated from February 1992 through February 1993 and 66 isolated from February 1995 through February 1996, to six fluoroquinolones and several other antibiotics were compared with those of 27 isolates obtained from 1981 through 1984. To identify mutations in gyrA and parC genes of the isolates, the quinolone resistance-determining regions of the gyrA and parC genes were PCR-amplified and the PCR products were directly sequenced. RESULTS: The minimum inhibitory concentration for 90% of strains (MIC90) values of norfloxacin for the isolates from 1992 to 93 (4 microg./ml.) and 1995 to 96 (8 microg./ml.) were 16- and 32-fold, respectively, higher than those for isolates from 1981 to 84 (0.25 microg./ml.). The MIC90 values of ciprofloxacin for isolates from 1992 to 93 (0.5 microg./ml.) and 1995 to 96 (1 microg./ml.) showed increase of 8- and 16-fold, respectively, in comparison with those from 1981 to 84 (0.063 microg./ml.). The isolates from 1992 to 93 and 1995 to 96 were also less susceptible to newer fluoroquinolones including levofloxacin, sparfloxacin, DU-6859a and AM-1155, as compared with those from 1981 to 84. In 46 (67.6%) and 16 (23.5%) of the 68 gonococcal strains sequenced, GyrA and ParC mutations were identified, respectively. No ParC substitutions were identified in any isolates without co-existence of the GyrA mutation. A Ser-91 to Phe mutation, which was detected in 30 (65.2%) of the 46 isolates with GyrA mutations, was the most common GyrA mutation. Mutants with the single Ser-91 to Phe substitution in GyrA were 12-fold and at least 13-fold, respectively, less susceptible to norfloxacin and ciprofloxacin than the wild type. CONCLUSIONS: The results obtained in this study suggest that a high prevalence of gonococcal isolates with the Ser-91 to Phe mutation in GyrA has reduced the susceptibility of this organism to fluoroquinolones in Japan.     

Contribution of mutations in gyrA and parC genes to fluoroquinolone resistance of mutants of Streptococcus pneumoniae obtained in vivo and in vitro

We have analyzed by gene amplification and sequencing mutations in the quinolone resistance-determining regions of the gyrA, gyrB, and parC genes of fluoroquinolone-resistant Streptococcus pneumoniae mutants obtained during therapy or in vitro. Mutations leading to substitutions in ParC were detected in the two mutants obtained in vivo, BM4203-R (substitution of a histidine for an aspartate at position 84 [Asp-84-->His]; Staphylococcus aureus coordinates) and BM4204-R (Ser-80-->Phe), and in two mutants obtained in vitro (Ser-80-->Tyr). An additional mutant obtained in vitro, BM4205-R3, displayed a higher level of fluoroquinolone resistance and had a mutation in gyrA leading to a Ser-84-->Phe change. We could not detect any mutation in the three remaining mutants obtained in vitro. Total DNA from BM4203-R, BM4204-R, and BM4205-R3 was used to transform S. pneumoniae CP1000 by selection on fluoroquinolones. For the parC mutants, transformants with phenotypes indistinguishable from those of the donors were obtained at frequencies (5 x 10(-3) to 8 x 10(-3)) compatible with monogenic transformation. By contrast, transformants were obtained at a low frequency (4 x 10(-5)), compatible with the transformation of two independent genes, for the gyrA mutant. Resistant transformants of CP1000 were also obtained with an amplified fragment of parC from BM4203-R and BM4204-R but not with a gyrA fragment from BM4205-R3. All transformants had mutations identical to those in the donors. These data strongly suggest that ParC is the primary target for fluoroquinolones in S. pneumoniae and that BM4205-R3 is resistant to higher levels of the drugs following the acquisition of two mutations, including one in gyrA.     

Interferons alpha/beta inhibit IL-7-induced proliferation of CD4- CD8- CD3- CD44+ CD25+ thymocytes, but do not inhibit that of CD4- CD8- CD3- CD44- CD25- thymocytes

We have determined partial sequences of the gyrA and parC genes of Enterobacter cloacae type strain including the regions analogous to the quinolone resistance-determining region of the Escherichia coli gyrA gene. The deduced 65- and 49-amino acid sequences of the determined regions of the E. cloacae gyrA and parC genes were identical to the corresponding regions of the E. coli GyrA and ParC proteins, respectively. We examined 40 clinical strains of E. cloacae isolated from patients with urinary tract infection for susceptibilities to nalidixic acid and ciprofloxacin. Based on the nalidixic acid and ciprofloxacin MICs, these isolates were divided into 19 quinolone-susceptible strains (MICs of nalidixic acid, 3.13-25 mg/L; MICs of ciprofloxacin, < or = 0.025 mg/L) and 21 quinolone-resistant strains (MICs of nalidixic acid, 400 to > 800 mg/L; MICs of ciprofloxacin, 0.39-100 mg/L). We analysed five quinolone-susceptible and 21 quinolone-resistant strains for alterations in GyrA and ParC. The five quinolone-susceptible strains had amino acid sequences in GyrA and ParC identical to those of type strain. Of the 21 quinolone-resistant isolates, three (MICs of nalidixic acid, 400 to > 800 mg/L; MICs of ciprofloxacin, 0.39-3.13 mg/L) had a single amino acid change at the position equivalent to Ser-83 in the E. coli GyrA protein and no alterations in ParC; one (MIC of nalidixic acid, > 800 mg/L; MIC of ciprofloxacin, 3.13 mg/L) had a single amino acid change at Ser-83 in GyrA and a single amino acid change at the position equivalent to Glu-84 in the E. coli ParC protein; two (MIC of nalidixic acid, > 800 mg/L; MIC of ciprofloxacin, 25 mg/L) had double amino acid changes at Ser-83 and Asp-87 in GyrA and no alterations in ParC; and 15 (MICs of nalidixic acid, > 800 mg/L; MICs of ciprofloxacin, 25-100 mg/L) had double amino acid changes at Ser-83 and Asp-87 in GyrA and a single amino acid change at Ser-80 or Glu-84 in ParC. This study suggests, that in clinical isolates of E. cloacae, DNA gyrase is a primary target of quinolones, that only a single amino acid change at Ser-83 in GyrA is sufficient to generate high-level resistance to nalidixic acid and to decrease susceptibility to ciprofloxacin, and that the accumulation of amino acid changes in GyrA and the simultaneous presence of the ParC alterations play a central role in developing high-level resistance to ciprofloxacin.     

Analysis of quinolone resistance mechanisms in a sparfloxacin-resistant clinical isolate of Neisseria gonorrhoeae

BACKGROUND AND OBJECTIVES: Recently, a reduction in the susceptibility of clinical isolates of Neisseria gonorrhoeae to newer fluoroquinolones including sparfloxacin in vitro has been recognized in Japan. The quinolone resistance mechanisms in gonococcal isolates from a patient with clinical failure of sparfloxacin treatment was investigated. GOAL: To report a man with gonococcal urethritis in whom clinical failure of sparfloxacin treatment occurred and to examine the quinolone resistance mechanisms in gonococcal isolates from the patient. STUDY DESIGN: A man with gonococcal urethritis was treated with oral 100 mg sparfloxacin three times daily for 5 days. However, clinical failure of the sparfloxacin treatment was observed. The antimicrobial susceptibilities of pretreatment and posttreatment isolates to sparfloxacin and other agents were measured. To analyze quinolone resistance mechanisms in the set of isolates, DNA sequencing of the genes corresponding to the quinolone resistance-determining regions within the GyrA and ParC proteins was performed. We also assayed the intracellular sparfloxacin accumulation level in these gonococcal cells. Moreover, we performed pulsed-field gel electrophoresis analysis to determine whether the pretreatment and posttreatment isolates were isogenic. RESULTS: The minimum inhibitory concentration of sparfloxacin for the posttreatment isolate (4 micrograms/ml) was 16 times higher than that for the pretreatment isolate (0.25 microgram/ml). The pretreatment isolate contained three mutations, including a Ser-91 to Phe mutation and an Asp-95 to Asn mutation in GyrA and a Ser-88 to Pro mutation in ParC. The posttreatment isolate had four mutations, including the same three mutations and an additional Glu-91 to Gly mutation in ParC. The sparfloxacin accumulation level within 30 minutes in the posttreatment isolate was four times less than that in the pretreatment isolate. There were no differences in the pulsed-field gel electrophoresis patterns between the pretreatment and posttreatment isolates from the patient. CONCLUSIONS: The emergence of a fluoroquinolone-resistant N. gonorrhoeae isolate with multiple mutations involving GyrA and ParC reduced the response to sparfloxacin treatment. Multiple dosing and long-term treatment with sparfloxacin seems to induce a mutation in ParC and an alteration leading to reduced drug accumulation that contribute to increasing the fluoroquinolone resistance level.     

Tumour size measured by transrectal ultrasonography in the staging of prostate cancer before radical prostatectomy

Fluoroquinolone-resistant mutants of Mycoplasma hominis were selected in vitro from the PG21 susceptible reference strain either by multistep selection on increasing concentrations of various fluoroquinolones or by one-step selection on agar medium with ofloxacin. The quinolone resistance-determining regions (QRDR) of the structural genes encoding the A and b subunits of DNA gyrase were amplified by PCR, and the nucleotide sequences of eight multistep-selected resistant strains were compared to those of susceptible strain PG21. Four high-level resistant mutants that were selected on norfloxacin or ofloxacin contained a C-to-T transition in the gyrA QRDR, leading to substitution of Ser-83 by Leu in the GyrA protein. Analysis of the sequence of the gyrB QRDR of the eight multistep-selected mutants did not reveal any difference compared to that of the gyrB QRDR of the reference strain M. hominis PG21. Similar analyses of eight one-step-selected mutants did not reveal any base change in the gyrA and gyrB QRDRs. These results suggest that in M. hominis, like in other bacterial species, a gyrA mutation at Ser-83 is associated with fluoroquinolone resistance.     

Detection of mutations in parC in quinolone-resistant clinical isolates of Escherichia coli

The gene parC encodes the A subunit of topoisomerase IV of Escherichia coli. Mutations in the parC region analogous to those in the quinolone resistance-determining region of gyrA were investigated in 27 clinical isolates of E. coli for which ciprofloxacin MICs were 0.0007 to 128 micrograms/ml. Of 15 isolates for which ciprofloxacin MICs were > or = 1 microgram/ml, 8 showed a change in the serine residue at position 80 (Ser-80), 4 showed a change in Glu-84, and 3 showed changes in both amino acids. No mutations were detected in 12 clinical isolates for which ciprofloxacin MICs were < or = 0.25 micrograms/ml. These findings suggest that ParC from E. coli may be another target for quinolones and that mutations at residues Ser-80 and Glu-84 may contribute to decreased fluoroquinolone susceptibility.     

Activities of new fluoroquinolones against fluoroquinolone-resistant pathogens of the lower respiratory tract

The activities of six new fluoroquinolones (moxifloxacin, grepafloxacin, gatifloxacin, trovafloxacin, clinafloxacin, and levofloxacin) compared with those of sparfloxacin and ciprofloxacin with or without reserpine (20 microg/ml) were determined for 19 Streptococcus pneumoniae isolates, 5 Haemophilus sp. isolates, and 10 Pseudomonas aeruginosa isolates with decreased susceptibility to ciprofloxacin from patients with clinically confirmed lower respiratory tract infections. Based upon the MICs at which 50% of isolates were inhibited (MIC50s) and MIC90s, the most active agent was clinafloxacin, followed by (in order of decreasing activity) trovafloxacin, moxifloxacin, gatifloxacin, sparfloxacin, and grepafloxacin. Except for clinafloxacin (and gatifloxacin and trovafloxacin for H. influenzae), none of the new agents had improved activities compared with that of ciprofloxacin for P. aeruginosa and H. influenzae. A variable reserpine effect was observed for ciprofloxacin and S. pneumoniae; however, for 9 of 19 (47%) isolates the MIC of ciprofloxacin was decreased by at least fourfold, suggesting the presence of an efflux pump contributing to the resistance phenotype. The laboratory parC (Ser79) mutant strain of S. pneumoniae required eightfold more ciprofloxacin for inhibition than the wild-type strain, but there was no change in the MIC of sparfloxacin and only a 1-dilution increase in the MICs of the other agents. For efflux pump mutant S. pneumoniae the activities of all the newer agents, except for levofloxacin, were reduced. Except for clinafloxacin, all second-step laboratory mutants required at least 2 microg of all fluoroquinolones per ml for inhibition.     

Inhibitory activities of quinolones against DNA gyrase and topoisomerase IV purified from Staphylococcus aureus

In order to clarify the mechanism of action of quinolones against Staphylococcus aureus, GrlA and GrlB proteins of topoisomerase IV encoded by genes with or without mutations were purified separately as fusion proteins with maltose-binding protein in Escherichia coli. The reconstituted enzymes showed ATP-dependent decatenation and relaxing activities but had no supercoiling activity. The inhibitory effects of quinolones on the decatenation activity of topoisomerase IV were determined by quantitative electrophoresis with kinetoplast DNA as a substrate. The 50% inhibitory concentrations (IC50s) of levofloxacin, DR-3354, DU-6859a, DV-7751a, ciprofloxacin, sparfloxacin, and tosufloxacin against topoisomerase IV of S. aureus FDA 209-P were 2.3, 97, 0.45, 1.5, 2.5, 7.4, and 1.8 microg/ml, respectively, and were correlated well with their MICs. The IC50s of these drugs were from 2 to 20 times lower than those for the DNA gyrase. These results support genetic evidence that the primary target of new quinolones is topoisomerase IV in quinolone-susceptible strains of S. aureus. Three altered proteins of topoisomerase IV containing Ser-->Phe changes at codon 80 or Glu-->Lys changes at codon 84 of grlA, or both, were also purified. The inhibitory activities of quinolones against the topoisomerase IV which contained a single amino acid change were from 8 to 95 times weaker than those against the nonaltered enzyme. These results suggest that the mutations in the corresponding genes confer quinolone resistance.     

Alterations in the GyrA subunit of DNA gyrase and the ParC subunit of DNA topoisomerase IV associated with quinolone resistance in Enterococcus faecalis

The gyrA and parC genes of 31 clinical isolates of Enterococcus faecalis, including fluoroquinolone-resistant isolates, were partially sequenced and analyzed for target alterations. Topoisomerase IV may be a primary target in E. faecalis, but high-level fluoroquinolone resistance was associated with simultaneous alterations in both GyrA and ParC.     

Gyrase mutations in laboratory-selected, fluoroquinolone-resistant mutants of Mycobacterium tuberculosis H37Ra

To characterize mechanisms of resistance to fluoroquinolones by Mycobacterium tuberculosis, mutants of strain H37Ra were selected in vitro with ofloxacin. Their quinolone resistance-determining regions for gyrA and gyrB were amplified and sequenced to identify mutations in gyrase A or B. Three types of mutants were obtained: (i) one mutant (TKp1) had no mutations in gyrA or gyrB; (ii) mutants that had single missense mutations in gyrA, and (iii) mutants that had two missense mutations resulting in either two altered gyrase A residues or an altered residue in both gyrases A and B. The TKp1 mutant had slightly reduced levels of uptake of [14C]norfloxacin, which was associated with two- to fourfold increases in the MICs of ofloxacin, ciprofloxacin, and sparfloxacin. Gyrase mutations caused a much greater increase in the MICs of fluoroquinolones. For mutants with single gyrA mutations, the increases in the MICs were 4- to 16-fold, and for mutants with double gyrase mutations, the MICs were increased 32-fold or more compared with those for the parent. A gyrA mutation in TKp1 secondary mutants was associated with 32- to 128-fold increases in the MICs of ofloxacin and ciprofloxacin compared with the MICs for H37Ra and an eight-fold increase in the MIC of sparfloxacin. Sparfloxacin was the most active fluoroquinolone tested. No sparfloxacin-resistant single-step mutants were selected at concentrations of > 2.5 micrograms/ml, and high-level resistance (i.e., MIC, > and = 5 micrograms/ml) was associated with two gyrase mutations. Mutations in gyrB and possibly altered levels of intracellular accumulation of drug are two additional mechanisms that may be used by M. tuberculosis in the development of fluoroquinolone resistance. Because sparfloxacin is more active in vitro and selection of resistance appears to be less likely to occur, it may have important advantage over ofloxacin or ciprofloxacin for the treatment of tuberculosis.     

Cloning and nucleotide sequences of the topoisomerase IV parC and parE genes of Mycoplasma hominis

The topoisomerase IV parC and parE genes from the wall-less organism Mycoplasma hominis PG21 were cloned and sequenced. The coupled genes are located far from the DNA gyrase genes gyrA and gyrB. They encode proteins of 639 and 866 amino acids, respectively. As expected, the encoded ParE and ParC proteins exhibit higher homologies with the topoisomerase IV subunits of the gram-positive bacteria Staphylococcus aureus and Streptococcus pneumoniae than with their Escherichia coli counterparts. The conserved regions include the Tyr residue of the active site and the region involved in quinolone resistance (quinolone resistance-determining region [QRDR]) in ParC and the ATP-binding site and the QRDR in ParE.     

Purification and in vitro reconstitution of the essential protein components of an aromatic polyketide synthase

The in vitro activities of seven quinolones and the sequences of the quinolone resistance-determining regions (QRDR) in the A and B subunits of DNA gyrase were determined for 14 mycobacterial species. On the basis of quinolone activity, quinolones were arranged from that with the greatest to that with the least activity as follows: sparfloxacin, levofloxacin, ciprofloxacin, ofloxacin, pefloxacin, flumequine, and nalidixic acid. Based on MICs, the species could be organized into three groups: resistant (Mycobacterium avium, M. intracellulare, M. marinum, M. chelonae, M. abscessus [ofloxacin MICs, >/=8 microg/ml]), moderately susceptible (M. tuberculosis, M. bovis BCG, M. kansasii, M. leprae, M. fortuitum third biovariant, M. smegmatis [ofloxacin MICs, 0.5 to 1 microg/ml]), and susceptible (M. fortuitum, M. peregrinum, M. aurum [ofloxacin MICs, 相似文献   

Constitutive activation of the TSH receptor by spontaneous mutations affecting the N-terminal extracellular domain

Activating mutations of the TSH receptor gene have been found in toxic adenomas and hereditary toxic thyroid hyperplasia. Up to now, all mutations have been located in the serpentine portion of the receptor. We now describe two additional mutations affecting Ser-281 (Ser-281-Thr and Ser-281-Asn) in the ectodomain of the receptor. After transfection in COS cells, both mutants displayed increased constitutive activity for cAMP generation despite expression at a lower level than the wild type. The mutants were responsive to TSH. The present results are compatible with a model in which the activity of the unliganded receptor is kept at a low level by an inhibitory interaction between the N-terminal domain and the serpentine portion of the receptor.     

Ectopic expression of inactive forms of yeast DNA topoisomerase II confers resistance to the anti-tumour drug, etoposide

Drug resistance to anti-tumour agents often coincides with mutations in the gene encoding DNA topoisomerase II alpha. To examine how inactive forms of topoisomerase II can influence resistance to the chemotherapeutic agent VP-16 (etoposide) in the presence of a wild-type allele, we have expressed point mutations and carboxy-terminal truncations of yeast topoisomerase II from a plasmid in budding yeast. Truncations that terminate the coding region of topoisomerase II at amino acid (aa) 750, aa 951 and aa 1044 are localised to both the cytosol and the nucleus and fail to complement a temperature-sensitive top2-1 allele at non-permissive temperature. In contrast, the plasmid-borne wild-type TOP2 allele and a truncation at aa 1236 are nuclear localised and complement the top2-1 mutation. At low levels of expression, truncated forms of topoisomerase II render yeast resistant to levels of etoposide 2- and 3-fold above that tolerated by cells expressing the full-length enzyme. Maximal resistance is conferred by the full-length enzyme carrying a mutated active site (Y783F) or a truncation at aa 1044. The level of phosphorylation of topoisomerase II was previously shown to correlate with drug resistance in cultured cells, hence we tested mutants in the major casein kinase II acceptor sites in the C-terminal domain of yeast topoisomerase II for changes in drug sensitivity. Neither ectopic expression of the C-terminal domain alone nor phosphoacceptor site mutants significantly alter the host cell's sensitivity to etoposide.     

Prevalence of a putative efflux mechanism among fluoroquinolone-resistant clinical isolates of Streptococcus pneumoniae

Twenty-three norfloxacin-selected first-step mutants of Streptococcus pneumoniae showed low-level fluoroquinolone resistance. Their susceptibility to norfloxacin in the presence or absence of reserpine and known efflux pump substrates was determined by an agar dilution method. Five mutants showed four- to eightfold increases in their susceptibility to norfloxacin in the presence of reserpine and four- to eightfold decreases in their susceptibility to acriflavine and ethidium bromide. This phenotype is suggestive of an efflux mechanism of resistance. A representative of these mutants, 1N27, accumulated significantly less ethidium bromide than the parent strain; reserpine abolished these differences. No changes in the quinolone resistance-determining regions of parC, parE, gyrA, or gyrB were found in this mutant. By our validated agar dilution method, the efflux phenotype was sought in clinical isolates of S. pneumoniae. Of 1,037 clinical isolates examined from the United Kingdom, 273 showed reduced susceptibility to norfloxacin or ciprofloxacin. Of these, 45.4% showed the efflux phenotype. Our findings suggest that an efflux mechanism may be a frequent cause of clinically significant fluoroquinolone resistance in pneumococci.